The present invention relates to integrin inhibitors useful for their ability to antagonize/block biological processes mediated by xcex1vxcex23 and related integrin receptors, to combinatorial and solid phase methods for preparing libraries of compounds, and utilization of libraries of the compounds for drug discovery. The present invention further provides pharmaceutical compositions for administration to mammals, including man, and methods for their use in the treatment of various disorders including, but not limited to, cancer (tumor metathesis, tumorgenesis/tumor growth), angiogenesis (as in cancer, diabetic retinopathy, rheumatoid arthritis), restenosis (following balloon angioplasty or stent implantation), inflammation (as in rheumatoid arthritis, psoriasis), bone diseases (osteopenia induced by bone metastases, immobilization and glucocortocoid treatment, periodontal disease, hyperparathyroidism and rheumatoid arthritis), and as antiviral agents.
The solid phase synthesis of non-peptidic small organic molecules is a rapidly evolving area of research with applications in the preparation of combinatorial libraries. While the solid phase synthesis of peptides is well established, the solid phase synthesis of non-peptidic small organic molecules is still evolving (Hermkens, P. H. H.; Ottenheijm, H. C. J.; Rees, D. Tetrahedron 1996, 52, 4527-4554). In particular, methods for solid phase synthesis of molecules of biological significance is of importance to drug discovery and is an active area of research.
The integrin xcex1vxcex23 has been shown to mediate the invasion of cancerous melanoma cells into healthy tissue and to protect these cells against natural cell death cycle (apoptosis). Vitronectin receptor(xcex1vxcex23) antagonists have been shown to inhibit the growth of various solid tumors of human origin. More recently, xcex1vxcex23 has been shown to be involved in liver metastasis. Although angiogenesis is an important and natural process in growth and wound healing, it is now appreciated that a variety of clinically relevant conditions are pathologically related to these processes, and that the integrin xcex1vxcex23 is involved. For example, xcex1vxcex23 was shown to be expressed on human wound tissue but not on normal skin and is preferentially expressed on angiogenic blood vessels, such as those feeding a growing/invading tumor. It has also been shown that antagonists of xcex1vxcex23 promote tumor regression by inducing apoptosis of the tumor cells. This process of neovascularization (new blood vessel growth, angiogenesis), which is critical for tumor growth and metastasis, is also an important event in occular tissue, leading to diabetic retinopathy, glaucoma and blindness and in joints, promoting rheumatoid arthritis.
xcex1vxcex23 has been shown to play a pivotal role in the proliferation and migration of smooth muscle and vascular endothetial cells, a pathological process leading to restenosis after balloon angioplastly (Choi et al., J. Vasc. Surgery, 1994, 19, 125-134; Matsumo et al., Circulation, 1994, 90, 2203-2206). At least one type of virus (adenovirus) has been shown to utilize (xcex1vxcex23 for entering host cells (White et al., Current Biology, 1993, 596-599).
Various bone diseases involve bone resorption-the dissolution of bone matter, which is mediated by only one known class of cells, the osteoclasts. When activated for resorption, these motile cells initially bind to bone, a process well known to be mediated by asps (Davies et al., J. Cell. Biol., 1989, 109, 1817-1826; Helfrich et al., J Bone Mineral Res., 1992, 7, 335-343). It is also well known that blockade of xcex1vxcex23 with antibodies or RGD containing peptides block osteoclast cell adhesion and bone resorption in vitro (Horton et al., Exp. Cell Res. 1991, 195, 368-375) and that echistatin, an RGD containing protein, inhibits bone resorption in vivo (Fisher et al., Endocrinology, 1993, 132, 1411-1413). More recently, an RGD peptidomimetic has likewise been shown to inhibit osteoclats in vitro and, by i.v. administration prevents osteoporosis (Engleman et al., J. Clin. Invest., 1997, 99, 2284-2292). Numerous patents/applications have claimed various non-peptide xcex1vxcex23 inhibitors for some or all of the above applications (e.g.WO 95/32710, WO 97/08145, WO 97/33887, U.S. Pat. No. 5,681,820).
Combinatorial chemistry is becoming an important tool for drug discovery and lead optimization (Borman, S. Chemical and Engineering News 1997, 75 (8), 43-63). A combinatorial synthesis requires that at least two components of the product molecules be independently variable, so that all of the combinations of these components can be prepared. A synthesis with three independently variable components is preferable since greater diversity in structure can be produced in the resultant library. Thus to prepare a combinatorial library of integrin inhibitors with a high degree of potential diversity and wide utility for drug discovery using solid phase techniques, it is important to identify a synthesis in which three components can be independently varied.
Most of the reported integrin inhibitors are RGD mimics and they use a xcex2-amino acid like substituted 2,3-diaminopropionic acid as the carboxylic acid terminus. While a cyclic or acyclic guanidino moiety is preferred for the basic end of the molecule, substituted ureas and amidines are used as well. The central scaffold, connecting these two pieces, itself can be varied widely. By developing a convenient route to appropriately protected fragments and a mild solid phase synthesis that incorporates all the three components in an independent fashion, it is possible to prepare combinatorial libraries of this important class of integrin inhibitors.
A solid-phase synthesis of integrin antagonist has been reported recently (Corbett, J. W.; Graciani, N. R.; Mousa, S. A.; DeGrado, W. F. Bioorganic and Med Chem Lett. 1997, 7, 1371-1376). However, this synthesis on solid phase does not provide a means of varying the substitutions on the xcex2-amino acid of the carboxy terminus and uses the commercially available xcex1-N-CBZ-diaminopropionic acid as the only fragment. Hence, a combinatorial library synthesized using this method has limited utility in the drug discovery process lacking structure-activity data for all the regions of the molecule that can be independently varied. It is important to optimize this region of the inhibitors since the lipophilic substitutents in this region and the linkers used to connect these substituents have a significant effect on the activity of this class of molecules.
Multiple compounds can be generated simultaneously by solid phase synthesis. The solid phase synthesis detailed in the present invention for the simultaneous generation of a library of integrin inhibitors where all three components can be varied is not known. The preparation of libraries of compounds of the present invention is useful because it provides rapid structural variation and structure-activity information.
Accordingly, the present invention discloses a solid phase synthesis process for producing compounds represented in formula (I): 
wherein: 
R1 and R2 independently are alkyl of 1-8 carbon atoms, alkenyl of 2-8 carbon atoms, alkynyl of 2-8 carbon atoms, cycloalkyl of 3-12 carbon atoms, aryl, aralkyl of 6 to 10 carbon atoms, heterocycloalkyl of 5-10 members consisting of carbon atoms and from 1 to 3 heteroatoms selected from N, S and O;
R3 is H, alkyl of 1-6 carbon atoms, aralkoxy of 1-6 carbon atoms;
X is NHCOO, NHCO, NHCONH, NHSO2;
Y is CH2, NH;
Z is CH, N, S
m is 0-4; and
n is 0-3; or pharmaceutical salts thereof.
In some aspects of the invention G may preferably be pyrimidinyl, guanidine, pyridyl-urea, benzyl-urea, azepinyl, imidazolinyl or tetrahydropyrimidinyl.
In other aspects of the invention R1 may be methyl, ethyl, n-propyl, i-propyl, allyl, homoallyl, propargyl, pentyl, n-hexyl, octyl, neopentyl, trichloroethyl, n-butyl, i-butyl, butynyl, phenyl, methylphenyl, dimethylphenyl, halophenyl, methoxyphenyl, acetylphenyl, biphenyl, naphthyl, benzyl, phenethyl, cyclohexyl, cyclohexylmethyl, trimethylcyclopropyl, phenylcyclopropyl, adamantyl, adamantylmethyl, cinnamic, pyridyl, or dimethylfuranyl.
In some preferred aspects of the present invention are provided compounds of Formula (I) wherein G, W, R1, R2, R3, X, Y, Z, m and n are defined above, with the proviso that when W is 
and R3 is H, then G is not: 
In still other preferred embodiments of the present invention G is 
In other embodiment of the present invention it is preferred that W is 
In yet other embodments of the present invention G is 
and W is 
xe2x80x9cAlkylxe2x80x9d, whether used alone or as part of a group such as xe2x80x9calkoxyxe2x80x9d, means a branched or straight chain having from 1 to 10 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl and hexyl. Lower alkyl refers to alkyl having from 1 to 6 carbon atoms. In some preferred embodiments of the present invention alkyl is from 1 to 8 carbon atoms. Alkyl groups may be substituted with one or more substituents selected from halogen, lower alkyl, lower alkoxy, lower alkylthio, amino, nitro, cyano, carboxy, alkylamino, perhaloalkyl, hydroxy, oxy, phenyl, phenylalkyl, or naphthyl.
xe2x80x9cCycloalkylxe2x80x9d as used herein refers to mono or polycyclic alkyl group of 3-12 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclohexyl and adamantyl. Cycloalkyl groups may be substituted. One preferred substitution is phenyl.
xe2x80x9cArylxe2x80x9d whether used alone or as part of a group such as xe2x80x9caralkylxe2x80x9d, means mono or bicyclic aromatic ring having from 5 to 10 carbon atoms. Exemplary aryl groups include phenyl and naphthyl. The aryl may be substituted with one or more substituents. Substituents include halogen, lower alkyl, lower alkoxy, lower alkylthio, amino, nitro, cyano, carboxy, carboxyalkyl, alkanoyl, alkylamino, perhaloalkyl, hydroxy, oxy, phenyl, phenylalkyl, or naphthyl. One preferred aryl group is phenyl which may be denoted as Ph in some instances.
xe2x80x9cHeterocycloalkylxe2x80x9d whether used alone or as part of a group such as xe2x80x9cheterocycloalkyl-alkylxe2x80x9d means a stable 5 to 10 membered mono or bicyclic ring of carbon atoms and from 1 to 3 heteroatoms selected from N, O and S. Exemplary heterocycloalkyls include pyrazinyl, pyrazolyl, tetrazolyl, furanyl, thienyl, pyridyl, imidazolyl, pyrimidinyl, tetrahydropyrimidinyl, isoxazolyl, thiazolyl, isothiazolyl, quinolinyl, indolyl, isoquinolinyl, oxazolyl and oxadiazolyl. Preferred heterocycloalkyl groups include pyrimidinyl, tetrahydropyrimidinyl, pyridyl, azepinyl, and imidazolyl. Most preferred heterocycloalkyls include pyridin-2yl, and tetrahydropyrimidine. The heterocycloalkyl may also be substituted with one or more substituents. Substituents include halogen, lower alkyl, lower alkoxy, lower alkylthio, amino, nitro, cyano, carboxy, carboxyalkyl, alkanoyl, alkylamino, perhaloalkyl, hydroxy, oxy, phenyl, phenylalkyl or naphthyl. Preferred substituents include amino and oxy. Preferred substituted heterocyloalkyls include 6 aminopyridin-2yl and tetrahydropyrimid-4-one.
xe2x80x9cAralkylxe2x80x9d means an aryl-alkyl group in which the aryl and alkyl are as previously described. Exemplary aralkyl groups include benzyl and phenethyl. Used in this context, the alkyl group may include one or more double bonds.
xe2x80x9cHeterocycloalkyl-alkylxe2x80x9d means an heterocycloalkyl-alkyl group in which the heterocycloalkyl and alkyl are as previously described. Used in this context the alkyl group may include one or more double bonds. Exemplary heterocycloalkyl-alkyls include pyridylmethyl, pyridylethyl, thienylethyl, thienylmethyl, indolylmethyl, and furylmethyl.
xe2x80x9cAlkoxyxe2x80x9d means an alkyl-O group in which the alkyl group is as previously described. Exemplary alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, and t-butoxy.
xe2x80x9cAralkoxyxe2x80x9d means an aryl-alkoxy group in which aryl and alkoxy are as previously described.
xe2x80x9cHalogenxe2x80x9d includes fluorine, chlorine, iodine and bromine.
xe2x80x9cProdrugxe2x80x9d, as used herein means a compound which is convertible in vivo by metabolic means (e.g. by hydrolysis) to a compound of Formula I.
Compounds of the present invention include all crystalline forms, pharmaceutically acceptable salts, enantiomers, racemic mixtures, and diasteromeric mixtures thereof.
Some preferred compounds of the present invention include:
2-benzyloxycarbonylamino-3-(2-{4-[2-(3-benzylureido)-ethyl]-piperazin-1-yl}acetylamino)-propionic acid; and
2-benzyloxycarbonylamino-3-(2-{4-[2-(3-benzylureido)-ethyl]-2,4-dioxo-3,4-dihydro-2H-quinazolin-1-yl}acetylamino)-propionic acid and pharmaceutically acceptable salts thereof
Compounds of the present invention may be prepared in accordance certain solid phase methodology. In one aspect of the present invention, compounds of Formula (I) where 
may be prepared in accordance with the steps of:
a) attaching a xcex2-amino acid of the formula 
xe2x80x83to a solid support P to produce a compound of formula (1) 
xe2x80x83wherein P is preferably a polystyrene resin cross-linked with divinylbenzene and functionalized with a linker such as a hydroxymethylphenoxy group, which is more preferably Wang""s resin:
b) deblocking the fluorenylmethyloxy carbonyl group of said compound of formula (1) with piperidine to produce a compound of formula (2); 
c) acylating compound of formula (2) with a chemical species selected from chloroformates, isocyanates, sulfonyl chlorides, carboxylic acid chlorides or carboxylic acids to produce a compound of formula (3) 
xe2x80x83wherein X and R1 are as defined above;
d) deblocking the 4,4-dimethyl-2,6-dioxocylohex-1-ylideneethyl protecting group of said compound of formula (3) with hydrazine to produce a compound of formula (4); 
e) reacting said compound of formula (4) with a Fmoc protected amino carboxylic acid of formula (5) 
xe2x80x83wherein W is as defined above, to produce a compound of formula (6); 
f) deblocking the fluorenylmethyloxy carbonyl group of said compound of formula (6) with piperidine to produce a compound of formula (7); 
g) reacting said compound of formula (7) with guanidilation reagents of formula (8) or (9) or (10) or amidation reagent of formula (11) 
xe2x80x83to produce a compound of formula (12) 
xe2x80x83and
h) reacting said compound of formula (12) with a cleaving reagent like trifluoroacetic acid to produce a compound of formula (I) 
xe2x80x83wherein R1, X, W and G are as defined above.
Also in accordance with the present invention, where 
compounds of Formula I may be prepared in accordance with steps a) through f) to produce compound of formula (7). This aspect of the methods of the invention further comprises the steps of:
i) reacting said compound of formula (7) with isocyanates or with p-nitrophenyl chloroformate, followed by amine to produce a compound of formula (12) 
xe2x80x83and
i) reacting said compound of formula (12) with a cleaving reagent like trifluoroacetic acid to produce a compound of formula (I) 
xe2x80x83wherein R1, X, W and G are as defined above.
The compounds of the present invention may be prepared according to the general process outlined in Scheme I. 
Thus, the orthogonally protected 2,3-diaminopropionic acid is attached to a solid support P, which is preferably a resin of polystyrene cross-linked with divinylbenzene and with a linker such as 4-hydroxymethylphenoxy, most preferably Wang""s resin as described below, in the presence of a coupling reagent such as 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/N-hydroxybenzotriazole (HOBT) to produce a compound of formula (1). The compound of formula (1) is deprotected using 20% piperidine in DMF to yield a compound of formula (2), which provides the first handle for diversification.
A compound of formula (2) is reacted with either chloroformates or isocyantes or carboxylic acid chlorides or sulfonyl chlorides in a solvent like dichloromethane or tetrahydrofuran to yield a compound of formula (3). Alternatively, in the case of amide formation, a carboxylic acid is coupled directly with the compound of formula (2) in the presence of a coupling reagent like 1,3-diisopropylcarbodiimide (DIC) to produce the compound of formula (3). A compound of formula (3) is treated with 2% hydrazine to deprotect the (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (dde) protecting group to yield a compound of formula (4), which provides a second handle for diversification. A compound of formula (4) is reacted with a Fmoc protected amino carboxylic acid of formula (5) in the presence of a coupling reagent like DIC to produce the compound of formula (6). The compound of formula (6) is deprotected using 20% piperidine in DMF to yield a compound of formula (7), which provides the third handle for diversification. The compound of formula (7) is reacted with N,N-bis-Boc-S-ethylthiourea or 2-(3,5-dimethylpyrazolyl)-4,5-dihydroimidazole or 2-bromopyrimidine or 1-methoxy-2-azacylohept-1-ene to give a compound of formula (12). Alternatively, the compound of formula (7) is reacted with p-nitrophenyl chloroformate and the resulting carbamate was reacted with amines to give ureas of formula (12).
The compounds of the present invention are integrin inhibitors useful for their ability to antagonize biological processes mediated by xcex1vxcex23 and related integrin receptors including, but not limited to, cancer (tumor metastatis, tumorgensis, tumor growth), angiogenesis (as in cancer, diabetic retinopathy, rheumatoid arthritis), restenosis (following balloon angioplasty or stent implantation), inflammation (as in rheumatoid arthritis, psoriasis), bone diseases (osteopenia induced by bone metastases, immobilization and glucocortocoid treatment, periodontal disease, hyperparathyroidism and rheumatoid arthritis), and as antiviral agents. The effect of the compounds to inhibit integrin is determined by standard pharmacological tests.
The purpose of this assay is to measure the effect of various compounds on the xcex1vxcex23-ligand interaction.
Reagents
Plasma Membrane Isolation: 15 confluent T150 flasks of 512P5 cells (xcex1vxcex23 -over expressing cell line) are washed 2xc3x97 with Dulbecco""s phosphate buffered saline (D-PBS) without calcium or magnesium, pH 7.1. Cells are harvested with 10 mL of trypsin/EDTA and collected by centrifugation. The cell pellet is washed 2xc3x97 with 0.5 mg/mL of soybean trypsin inhibitor, and resuspended at 10% weight/volume in homogenization buffer (25 mM Tris-HCl, pH=7.4; 250 mM sucrose). The cell suspension is homogenized with 2xc3x9730(seconds bursts of a Polytron homogenizer. The homogenate is centrifuged at 3000 g for 10 minutes at 4xc2x0 C. The supernatant is collected, measured, and made 100 mM in NaCl and 0.2 mM in MgSO4. The supernatant is centrifuged at 22,000 g for 20 minutes at 4xc2x0 C., the pellet is resuspended in 7 mL of membrane buffer (25 mM Tris-HCl, pH=7.4; 100 mM NaCl; 2 mM MgCl2) by 5 strokes of a Dounce homogenizer (tight pestle) and recentrifuged at 22,000 g for 20 minutes at 4xc2x0 C. The pellet is resuspended in 0.5 mL/flask of membrane buffer (stock membranes) and frozen at xe2x88x9280xc2x0 C. Prior to use, stock membranes are Dounce homogenized and diluted 2 xcexcL to 1000 xcexcL in membrane buffer. See References
Compound Dilution: The stock compounds are dissolved in an appropriate vehicle (typically DMSO) and subsequently diluted in assay buffer composed as follows: 25 mM Tris-HCl (pH=7.4), 100 mM NaCl, 2 mM MgCl2, 0.1% BSA.
Plate Preparation
Wells of Multiscreen-FB assay plates (Millipore MAFB NOB 50) are blocked with 150 mL of 0.1% polyethylenimine for 2 hours at 4xc2x0 C. Following incubation the wells are aspirated and washed with isotonic saline solution.
Binding Assay
125 xcexcL of assay buffer is added to each well. Next, 25 xcexcL of labeled ligand is added to each well. 25 xcexcL of unlabeled ligand is added to non-specific binding wells (NSB). 25 xcexcL of assay buffer is added to all other wells. 2 xcexcL of compound is added to appropriate sample wells, and 2 xcexcL of DMSO is added to NSB and total binding (TB) wells. Finally, 25 xcexcL of membrane is added to each well.
The plates are covered and incubated at 37xc2x0 C. for 2 hours in a humidified incubator. Wells are aspirated on a Millipore vacuum manifold, and the wells are washed with 150 xcexcL isotonic saline solution. Wells are again aspirated. The plates are then dried for 1 hour in an 80xc2x0 C. vacuum drying oven. Plates are placed on a Millipore filter punch apparatus, and filters are placed in 12xc3x9775 mm polypropylene culture tubes. The samples are counted on a Packard gamma counter.
Using 125I-Echistatin (specific activity=2000 Ci/mmol) supplied by Amersham at a final concentration of 50 pM, the following parameters are routinely observed;
Analysis of Results
The individual well activity is expressed as a percentage of the specific binding; % Max, and reported as the meanxc2x1standard deviation. Dose-inhibition relationships are generated for dose (X-axis) vs. % Max (Y-axis) for active compounds using a non-linear regression computer program (PS-NONLIN), and IC50 values with corresponding 95% confidence intervals are estimated from 50% of maximal attachment.
Reference Compounds
Various Arginine-Glycine-Aspartic Acid (RGD)-containing peptides were assessed for the ability to inhibit avb3 binding and the corresponding IC50 values with 95% confidence intervals were generated; peptide structures are given by the standard single letter designation for amino acids. Values obtained compared favorably with adhesion assay results.
1. Nesbitt, S. A. And M. A. Horton, (1992), A nonradioactive biochemical characterization of membrane proteins using enhanced chemiluminescence, Anal. Biochem., 206 (2), 267-72.
The purpose of this assay is to measure the effect of various compounds on the RGD-dependent attachment of cells to osteopontin mediated by the xcex1vxcex23 integrin.
Reagents
Cell Suspension Media
The cells are suspended for assay in the tissue culture media used for normal culture maintenance buffered with 25 mM HEPES (pH 7.4) without serum supplementation.
Compound Dilution Media
The stock compounds are dissolved in an appropriate vehicle (typically DMSO) and subsequently diluted in the tissue culture media used for normal culture maintenance buffered with 25 mM HEPES (pH 7.4) supplemented with 0.2% BSA (no serum); final vehicle concentration is xe2x89xa60.5%.
Plate Preparation
Human recombinant osteopontin (Structural Biology Group, W-AR) is diluted to an appropriate concentration in Dulbecco""s phosphate buffered saline (D-PBS) without calcium or magnesium, pH 7.1. 100 mL of this solution is incubated in the wells of PRO-BIND assay plates (Falcon 3915) for 2 hours at 37xc2x0 C. Following incubation the wells are aspirated and washed once with D-PBS; plates can either be used immediately or stored for up to 1 week at 4xc2x0 C. Prior to assay, the wells are blocked with 1% bovine serum albumin (BSA) in cell suspension media for 1 hour at 37xc2x0 C. Following the blocking period, wells are aspirated and washed once with D-PBS.
Cell Suspension
xcex1vxcex23-expressing cell lines are maintained by standard tissue culture techniques. For assay, the cell monolayer is washed three times with D-PBS, and the cells are harvested with 0.05% trypsin/0.53 mM EDTA (GIBCO). The cells are pelleted by low-speed centrifugation and washed three times with 0.5 mg/mL trypsin inhibitor in D-PBS (Sigma). The final cell pellet is resuspended in cell suspension media at a concentration of 106 cells/mL.
Attachment Assay
Incubation
100 mL of diluted test compound is added to osteopontin-coated wells (in triplicate) followed by 100 mL of cell suspension; background cell attachment is determined in uncoated wells. The plate is incubated at 25xc2x0 C. in a humidified air atmosphere for 1.5 hours. Following the incubation period, the wells are gently aspirated and washed once with D-PBS.
Cell Number Detection
The number of cells attached is determined by an MTT dye conversion assay (Promega) according to the manufacturer""s instructions. Briefly, MTT dye is diluted in cell suspension media (15:85) and 100 mL is added to each well. The assay plates are incubated for 4 hours at 37xc2x0 C. in a humidified 5% CO2/95% air atmosphere, followed by the addition of 100 mL stopping/solubilization solution. The assay plates are covered and incubated at 37+ C. in a humidified air atmosphere overnight. After the solubilization period, the optical density of the wells is measured at a test wavelength of 570 nM with a reference measurement taken simultaneously at 630 nM.
Analysis of Results
The individual well optical density is expressed as a percentage of the maximal attachment (% Max) wells minus background attachment, and reported as the meanxc2x1standard deviation. Dose-inhibition relationships are generated for dose (X-axis) vs. % Max (Y-axis) for active compounds using a non-linear regression computer program (PS-NONLIN), and IC50 values with corresponding 95% confidence intervals are estimated from 50% of maximal attachment.
Reference Compounds
Various Arginine-Glycine-Aspartic Acid (RGD)-containing peptides, and monoclonal antibodies were assessed for the ability to inhibit osteopontin-xcex1vxcex23 attachment and the corresponding IC50 values with 95% confidence intervals were generated in the SK-MEL-24 human malignant melanoma cell line; peptide structures are given by the standard single letter designation for amino acids:
Ruoslahti, R. Fibronectin and its receptors. Ann. Rev. Biochem. 57:375-413, 1988.
Hynes, R. O. Integrins: Versatility, modulation, and signaling in cell adhesion. Cell. 69: 11-25, 1992.
Osteoclast Pitting Assay
The assay is conduct ed as described in Murrills and Dempster (1990) Bone 11:333-344. Briefly, 4xc3x974xc3x970.2 mm slices of devitalized bovine cortical bone are numbered, placed in the wells of 96-well culture plates and wetted with 100 ul of Medium 199 containing Hanks salts, 10 mM HEPES, pH 7.0 (Medium 199/Hanks). Bone cell suspensions containing osteoclasts are prepared by mincing the long bones of neonatal rats (Sprague-Dawley , 4-6 days old) in Medium 199/Hanks. 100 uL of the suspension are then plated onto each slice and incubated 30 minutes to allow osteoclasts to adhere. The slices are rinsed to remove non-adherent cells and incubated 24 h in Medium 199 containing Earle""s salts, 10 mM HEPES and 0.7 g/L NaHCO3, which equilibrates at pH 6.9 in a 5% CO2 atmosphere. At this pH the adherent osteoclasts excavate an adequate number of resorption pits for assay purposes. Slices are fixed in 2.5% glutaraldehyde and osteoclasts counted following tartrate-resistant acid phosphatase staining. In experiments in which osteoclast numbers are significantly reduced in a particular treatment, a check is made for non-specific cytotoxicity by counting the number of contaminant fibroblast-like cells following toluidine staining. All cells are stripped from the slice by sonication on 0.25M NH4OH and the resorption pits formed by the osteoclasts during the experiment stained with toluidine blue. Resorption pits are quantified by manually counting.
Statistics
The experiments are conducted according to a block design with osteoclasts from each animal exposed to each treatment. Three replicate slices are used per treatment per animal, such that a total of 96 slices are examined for an experiment involving four animals and eight treatments (including control). Several parameters are recorded on a xe2x80x9cper slicexe2x80x9d basis: number of pits, number of osteoclasts, number of pits per osteoclast, number of fibroblast-like bone cells. SAS or JMP statistical software are used for statistical analysis. If analysis of variance reveals significant effects in the experiment, those treatments differing significantly from control are identified using Dunnett""s test. IC50s are calculated for active compounds using dose-response curves.
Reference Compound: Rat Calcitonin
Clinical Relavence
Osteoclasts are responsible for the bone loss that occurs in the onset of osteoporosis and anti-resorptive drugs directed against the osteoclast are a requirement for patients losing bone. Calcitonin and bisphosphonates, both used as anti-resorptives in the clinic, show significant osteoclast inhibitory activity in this assay. Hence it is a reasonable assay in which to identify novel anti-resorptives.